Process for preparing purified syphilis antigen from Treponema palljdum

ABSTRACT

A process for preparing a purified syphilis antigen from Treponema pallidum is presented. The process comprises the steps of obtaining an extract from Treponema pallidum, adsorbing the extract onto hydroxyapatite gel and eluting the antigen in the presence of a surfactant. The preferred surfactant is octylglucopyranoside, A diagnostic agent is prepared which comprises the purified syphilis antigen adsorbed on an inert carrier, which carrier at least partially a hydrophobic carrier to which the antigen is adsorbed.

This application is a continuation of U.S. application Ser. No.07/985,346 filed Nov. 30, 1992, now abandoned, is a continuation of U.S.application Ser. No. 07/704,526 filed May 24, 1991, now abandoned, whichis a continuation-in-part of Ser. No. 07/669,479 filed Mar. 14, 1991,abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for preparing an antigen ofTreponema (Treponema pallidum, hereinafter sometimes abbreviated to TP)which is used as a reagent for diagnosing syphilis. More particularly,the present invention relates to a method for preparing an antigen whichenables to prepare a diagnostic agent for syphilis, exhibiting highspecificity and being able to detect primary syphilis. Further, thisinvention relates to a diagnostic reagent for syphilis and a method forpreparing the same.

2. Prior Arts

Diagnostic methods have been performed which utilize theantigen-antibody reaction of TP antigens and anti-treponemal antibodies(hereinafter abbreviated to TP antibody) in sera from syphiliticpatients. Among such methods, TPHA (Treponema pallidum hemaggultinationassay test) has been widely used in recent years because of theadvantages in its sensitivity, specificity and convenience in operation.Therefore, the TPHA has been a typical diagnostic method for syphilis.

The antigen solution originated from TP and used in the above-mentionedmethod is prepared as follows: First, TP is inoculated and cultivated inrabbit testes. The treponemes are extracted and suspended in a suitablebuffer and then disrupted by homogenizer, sonicator and so forth. Thusdisrupted treponemes with or without solubilization was used as theantigen solution for sensitization.

However, the prior art has the following drawbacks. Specifically,primary syphilis can not sufficiently be detected by the diagnosticagent for syphilis made from the conventional TP antigen solution. Inother words, the conventional TPHA test or the like does not show apositive result in most cases until 2 to 3 months after syphiliticinfection. Accordingly, there is a great problem that, in order toaccomplish reliable diagnosis for primary syphilis, a diagnostic reagentusing a lipoidal antigen (cardiolipin) should be used together with theTPHA method. Although the reagent using lipoidal antigens is sensitiveto primary syphilis, nonspecific reactions are often observed.

In a syphilitic antibody detection test such as TPHA, sensitivity ofreagent to a primary antibody (Ig-M) is lower than an advanced antibody.This was caused by impurities in the antigen solution used for thereagent. Namely, in the conventional TPHA, the TP antigen solution usedfor sensitization of animal erythrocytes inevitably includes impuritiesdue to the preparation method. 90% or more of impurities are proteinsoriginated from rabbit testes in which TP is cultivated, or from TPcomponents having no antigenicity. Consequently, a significant quantityof impurities is incorporated in the TP antigen solution, so that theprimary antibody (Ig-M) cannot be detected.

The antigen solution used for the conventional TPHA inevitably includescomponents originated from rabbit tissue due to its preparation method,which cause nonspecific reaction. Therefore, in order to reduce thenonspecific reaction, some components originated from rabbit tissue wereadded to the buffer of TPHA for absorbing heterophil antibodies in serumto be tested.

In order to solve the above-mentioned problems, Japanese UnexaminedPatent Application No. SHO 58(1983)-71457 discloses a technique using anantigen fraction which is obtained by removing fractions having specificgravity of 1.01 or less from the extracted treponemal suspension.

However, a significant quantity of impurities is still mixed in theantigen fraction, because the fractionation method by the difference ofspecific gravity does not provide a strict separation of the antigenfraction from rabbit tissue. The aforesaid Japanese Application statesthat the above method is applicable even after the disruption oftreponemes. However, sodium diatrizoate or the like used for densitygradient reagents is inevitably incorporated in the antigen solution inthis method, and hence, a process for removing the density gradientreagents should be required. Accordingly, this method would not beapplied after the disruption of treponemes.

An immunological diagnostic reagent is generally prepared byimmobilizing an antigen or antibody on a hydrophobic carrier (e.g.,plastic particles such as latex particles, cellulose powder,polystyrene, polypropylene or nylon particles; membrane ofnitro-cellulose or nylon; erythrocytes treated with tannic acid; oragarose gel). Known immobilization methods include a method by physicaladsorption wherein an antigen or antibody is in contact with ahydrophobic carrier in an aqueous medium and a method wherein an antigenor antibody is covalently bonded to a carrier having an amino group or acarboxylic group on its surface. The former method utilizing physicaladsorption is widely used in view of manufacturing efficiency,convenience and being easy to reproduce the product of the same quality.

In the case of immobilizing an antigen or antibody by physicaladsorption, an approximately neutral buffer comprising a salt and abuffering agent is usually used as the aqueous medium. A surfactant isnot employed as the aqueous medium, since the surfactant is consideredto interfere with the immobilization of antigen or antibody on carrier.Specifically, the surfactant is considered to decrease the hydrophobicinteraction in immobilization by physical adsorption.

The surfactant is also considered to interfere with the immobilizationof TP antigens on carriers. In other words, the efficiency ofimmobilization is substantially decreased in the presence of thesurfactant, with the result that it is difficult to prepare an excellentdiagnostic reagent.

SUMMARY OF THE INVENTION

The present invention is accomplished to solve the problem of impuritiesin the antigen immobilized on the carrier in the method for preparingthe antigen originated from T. pallidum. A main object of the presentinvention is to provide a process for preparing a treponemal antigenused for a diagnostic agent of syphilis which can detect a primarysyphilis as well as an advanced syphilis and does not exhibit anonspecific reaction.

Thus, the present invention provides a process for preparing atreponemal antigen which comprises adsorbing an extract originated fromT. pallidum on a hydroxyapatite gel, followed by elution, while anaqueous medium is used.

Further, the present invention provides a diagnostic agent employing atreponemal antigen, preferably the above-mentioned one and a method forpreparing the same.

The diagnostic agent of syphilis in the present invention comprises atreponemal antigen and a carrier. Preferably, the agent is prepared bytreating a carrier with a treponemal antigen in an aqueous medium havinga pH from about 4.5 to about 7.7 and containing a surfactant in anamount of from about 0.01 wt. % to about 2.5 wt. % and removing theremaining surfactant if any.

PREFERRED EMBODIMENT OF THE PRESENT INVENTION

The extract originated from T. pallidum can be prepared in accordancewith the present invention as follows.

(1) Selection, cultivation and collection of T. pallidum

Suitable seed strain of T. pallidum is, for example, WHO's pathogenicstandard Nichols strain or T. pallidum strain used for diagnosingsyphilis in various tests. The WHO's pathogenic standard Nichols strainis easily available from, for example, CDC (Center for Disease Control,Public Health Service, U.S. Department of Health, Education and Welfare,Atlanta Ga.). The cultivation, collection and treatment methods canoptionally be selected among any known methods.

(2) Disruption and solubilization of TP

Subsequently, the collected treponemes are suspended in a buffersolution. After cooling and disruption, the resultant is solubilized toobtain the extract originated from TP. The method for disruption orsolubilization is optionally selected among any known methods. Thedisruption can be conducted by a homogenizer, ultrasonication orfreeze-thawing method. The solubilization can be performed with asurfactant for solubilization of slightly soluble protein, chaotropicion (e.g., SCN⁻, Cl⁻ or I⁻), urea or an alkaline treatment, an enzymatictreatment, an autolysis method or the like. Particularly, thesolubilization method is more suitable which employs a now ionicstrength buffer containing a non-ionic or amphoteric surfactant.

Besides, the present method is applicable to an extract containing TPantigen which is obtained from Escherichia coli or the like inaccordance with a recombinant DNA method, as far as said antigen has thephysicochemical characteristics existing within the range of those ofthe antigen of the present invention.

(3) Pretreatment

It is preferable that any pretreatment is carried out for removingimpurities beforehand as much as possible from the extract originatedfrom TP in order to effectively perform the present invention. ofcourse, the present invention can be carried out without thepretreatment.

Examples of the pretreatment are as follows:

(a) The fractions other than the antigen fraction are removed beforehandby an ion-exchange chromatography (utilizing, for example, a cationexchanger).

(b) A partial purification is conducted with a sodium sulfate orpolyethylene glycol fractination.

The above pretreatment (a) is more desirable since it is easier toperform compared with (b).

A preferable embodiment will be explained hereinbelow for performing themethod for preparing the treponemal antigen according to the presentinvention. It is to be noted that the present invention should not belimited to the embodiment described below.

(1) Hydroxyapatite gel

The hydroxyapatite used in the present invention can be represented bythe chemical formula of [Ca₅ (PO₄)₃ (OH)]₂. Usable gels are those onmarket, for example, Bio-Gel® HTP (Bio-Rad Laboratories) or HCA-200 L(Mitsui Toatsu Chemicals, Inc.). There is no limitation in the shape andthe particle size of the gel. Generally, gels for column chromatographycan be used. Preferably, the specific surface area of the gel is about 1to 100 m² /g and the particle size thereof is about 1 to 100 μm.

(2) Kind of the aqueous medium

Any buffer used for general biochemical experiment such as phosphatebuffer, Tris buffer, glycine buffer or the like can be used as theaqueous medium in the present invention. Phosphate buffer is preferable.

(3) pH of the aqueous medium

(a) Adsorption of the antigen

The pH of the aqueous medium is preferably within the range from about5.0 to 8.0, more preferably within the range from about 5.5 to 7.0. Whenthe pH is less than 5.0, antigenicity is likely to be lost, therebydecreasing recovery of the TP antigen. On the other hand, the aqueousmedium having pH 8.0 or more causes the reduction in the adsorptionefficiency of the antigen to the hydroxyapatite gel, thereby decreasingrecovery of the TP antigen.

(b) Elution of the antigen

The pH is preferably within the range from about 5.0 to 11.0, preferablyabout 5.5 to 10.0 for eluting the antigen. When the buffer has pH 8.0 ormore, its ionic strength is preferably to be in a low ionic strengthsuch as 10 to 60 mM to reduce the affinity of antigen for the column.When the pH is less than 5.0, antigenicity is likely to be lost, thusundesirable. On the other hand, an aqueous medium having a pH more than11.0 tends to elute the impurities together with the antigen, thusundesirable.

(4) Ionic strength of the aqueous medium

(a) Adsorption of the antigen

An aqueous medium having an ionic strength of preferably about 2 to 30mM, more preferably about 10 to 20 mM is used to adsorb the extractoriginated from TP on the hydroxyapatite gel.

When the ionic strength is less than 2 mM, it is not practical becauseimpurities tend to be adsorbed on the hydroxyapatite gel and thecapacity of the buffer used is reduced. On the other hand, when theionic strength is more than 30 mM, it causes less adsorption of theantigen on the hydroxyapatite gel, thereby decreasing the recovery.

(b) Elution of the antigen

The ionic strength in the elution is preferably about 10 to about 360mM, more preferably about 50 to about 120 mM. When the ionic strength isless than 10 mM, the antigen is hard to be eluted at an acidic pH. Theimpurities are also eluted with an ionic strength of more than 360 mM,thus undesirable. In case where the elution is conducted with an ionicstrength below that in the adsorption, the elution is preferably carriedout at a pH higher than that in the adsorption. On the other hand, theelution is preferably carried out with an ionic strength higher thanthat in the adsorption in case where the elution is conducted with a pHbelow that in the adsorption.

(5) Additive or the like to the aqueous medium

Any materials to which the hydroxyapatite gel is resistant such as thesurfactant, chaotropic ions or urea which can be used for solubilizationof treponemal antigen can be used as the additive to the aqueous mediumfor the adsorption and elution. A buffer of low ionic strength whichcontains a non-ionic or amphoteric surfactant is preferably used for themethod of the present invention in consideration of easiness ofoperation. Besides, the use of chelating agent such as EDTA is notrecommended since it interferes with the adsorption of the antigenprotein to the hydroxyapatite gel.

(6) Gradient elution

The elution can be carried out with stepwise or linear increase of theionic strength or pH, although the increasing pattern is notparticularly limited.

The stepwise increasing method would be industrially effective, becausethe antigen can be once eluted under a predetermined suitable condition.However, in such method, it is noted that the perfect separation of theantigen from impurities may not be attained in some cases. Accordingly,the linear gradient elution method which linearly increases the ionicstrength and/or pH is preferable. By this method, the optimal fractioncan be obtained by examining the elution profile on chromatogram, ordividing the fractions as detailedly as possible and measuring theantigen activity thereof.

By the present method, the substantially pure antigen can be obtained.

When the extract originated from treponemes is in contact with thehydroxyapatite gel in the buffer having a low ionic strength (saltconcentration), the antigens are adsorbed on the gel. Then, the antigensare eluted from the gel at the ionic strength within the predeterminedrange, thereby obtaining the antigen of high-purity.

Explained next is the application of the antigen obtained by the presentinvention.

(1) Diagnostic agent and a method for preparing the same

The treponemal antigens of the present invention are immobilized on acarrier by any known method to make a reagent for diagnosing syphilis.

Any carrier used in this field can be used. Preferable carriers areinert carriers in which the surface is at least partially hydrophobic.Examples of the carriers are synthetic polymer particles having aparticle size of about 0.05 to 50 μm prepared by performingpolymerization or copolymerization with a monomer such as styrene,acrylic acid, methyl methacrylate, acrylonitrile or butadiene andparticularly microparticles having an uniform particle size of 0.1 to 2μm, i.e., latex particles called in the field of an immunologicaldiagnostic reagent, prepared by emulsion-polymerizing the aforesaidpolymer or its derivatives in an aqueous medium; synthetic polymermaterials such as polystyrene, polyethylene, polypropylene, nylon orcellulose acetate and their molded product; membrane of nylon,nitrocellulose or the like; materials of living organism such as sheepor hen erythrocytes treated with tannic acid; and inorganic materialssuch as silica powder or glass particles, or the like. The latexparticles and erythrocytes treated with tannic acid are more preferable.

"The surface is at least partially hydrophobic" means the property forimmobilizing the antigen via any physical adsorption. The surface mayentirely be hydrophobic. Some materials may be used by activating thesurface thereof.

According to the present invention, it is found that the immobilizationof the antigen on a carrier is conducted in contacting them in anaqueous medium containing about 0.01 to 2.5% by weight of a surfactantand having pH about 4.5 to 7.7, thereby providing a reagent withexcellent sensitivity and improved specificity in which the antigens aremuch stably immobilized.

Besides, in case where the surfactant is used at a high concentration,it is preferable to remove the possibly remaining surfactant after theimmobilization, in order to avoid the interference of the surfactantwith the antigen-antibody reaction.

Usable surfactants for the above immobilization are those which can beused for extraction and stabilization of the surface antigen andmembrane protein of TP, are capable of extracting and solubilizing theobject constituents, have high specificity of extractability, and arestable at pH about 4.5 to 7.7 without separation. Preferable examples ofthe surfactants are non-ionic surfactants such as octylglucopyranoside(1-O-n-octyl-β-D-glucopyranoside), Triton X-100®, Tween 20®, Tween 80®,octylthioglucoside or the like or ampholytic surfactants such as CHAPS(3-[(3-Cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate) or thelike. Cationic surfactants such as dodecylamine or anionic surfactantssuch as sodium dodecyl sulfate can be used.

The effective concentration of the surfactant in the aqueous medium forimmobilizing the TP antigen on the carrier is about 0.01 to 2.5 wt. %,preferably about 0.02 to 2.10 wt. %. If the concentration of thesurfactant is higher, the TP antigen cannot be immobilized on thecarrier. The activity of the TP antigen is likely to be lost with thesurfactant of lower concentration.

Preferable examples of the aqueous media used for immobilizing the TPantigen on the carrier are buffers used for general biochemicalexperiments, e.g., phosphate buffer, Tris buffer or the like. The ionicstrength is adjusted by the addition of salt to the aqueous medium. Theeffective pH is about 4.5 to 7.7, preferably about 4.9 to 7.1, morepreferably about 5.4 to 6.5. If the pH is less than 5.0, antigenicity islikely to be lost, but the adsorption is performed instantaneously.Thus, the pH may be more than 4.5. The quantity of the TP antigenimmobilized on the carrier becomes less with the aqueous medium of pHmore than 7.7. In order to increase the storage stability of theobtained diagnostic reagent, a preservative can be added thereto ifnecessary.

Further, choline chloride, EDTA, saccharides (polysaccharide, dextran orthe like), polyethylene glycol and the like can be added in order toimprove the sensitivity in measurement.

Explained subsequently is a specific method for treating the carrierwith the antigen solution.

First, the antigen solution for immobilization is prepared by any knownmethod. For example, the treponemes are extracted from rabbit testicularmaterials containing treponemes. Then, the treponemes are washed, towhich a surfactant is added. The resultant mixture is incubated todisrupt the treponemes and extract the TP antigen. The extract iscentrifuged to collect the supernatant which is then diluted with abuffer containing a surfactant to obtain treponemal antigen solution.The obtained solution is adjusted to have a predetermined surfactantconcentration and pH, thus making the sensitizing solution, i.e., theantigen solution for immobilization.

Subsequently, the sensitizing solution is in contact with the carrierdescribed above in an aqueous medium containing about 0.01 to 2.5 wt. %of surfactant and having pH about 4.5 to 7.7. The resultant mixture wasincubated for a predetermined period to immobilize the TP antigen on thecarrier.

Other methods for immobilizing the TP antigen on the carrier includevarious ones, e.g., a method in which an antigen solution containing asuitable concentration of surfactant is added to a carrier suspensioncontaining or not containing surfactant to adjust the concentration ofsurfactant and pH to a predetermined value, and then results inimmobilization of the antigens, or a method in which a carrier iscontacted with an antigen solution and then a diluent comprising asurfactant or buffer is added to adjust the concentration of surfactantand pH to a predetermined value thereby immobilizing the TP antigen.

After the immobilization, the carrier is separated from the aqueousmedium and then washed with a buffer containing bovine serum albumin,saline solution or the like to remove the surfactant.

The antigen sensitizing solution described above is prepared from aso-called partially purified antigen fraction. An excellent diagnosticreagent can be obtained as mentioned below by using the treponemalantigen purified according to the present invention with the carrier oflatex particles.

The surfactant used for immobilizing the antigen on the carrier in thepresent invention is considered to stabilize the antigens in the antigensolution and prevent unnecessary components in the material from beingimmobilized on the carrier. In general, the surfactant interferes withthe immobilization of antigens on carriers. The antigens of the presentinvention are slightly soluble in water and hence low isoelectricpoints, whereby the solubility of the antigen is decreased by loweringpH so that the immobilization on carrier occurs even in the presence ofthe surfactant. As a result, the dignostic reagent of the presentinvention has an increased amount of the antigen carried on the carrierand more excellent sensitivity compared with that prepared by theconventional method. Further, amount of other components immobilized thecarrier decreases, thereby improving specificity.

(2) Application of the diagnostic reagent

The disgnostic agent of the present invention is used for detectinganti-treponemal antibodies in the subject serum. The detection methodsinclude radio-immunoassay (RIA), fluorescent immunoassay (FIA), enzymeimmunoassay (EIA or ELISA), latex agglutination assay, TPHA method(Treponema pallidum hemaggultination assay test) or the like. Thediagnostic reagent of the present invention can be formed to suit theabove-mentioned detection methods.

The immunoassay utilizing the antigen-antibody reaction is preferablyconducted in the presence of a water-soluble polymer and/orwater-soluble copolymer containing at least one kind of glycosidederivatives represented by the following formula (I) as a monomer:##STR1## wherein G--O-- represents a saccharic residue not having aprotective group. R is a hydrogen atom, a methyl group or an ethylgroup, m is an integer of 1 to 3 and n is an integer of 1 to 4.

The saccharic residue in the above glycoside derivative is a group inwhich a hydrogen atom is removed from a hydroxyl group bonded to theglycosidic carbon atom of the reduced end of a saccharide. Specifically,the saccharic residue means a residue of a monosaccharide comprising 1to 3 sugar units or an oligosaccharide.

Examples of the monosaccharides are hexoses such as glucose, mannose,galactose, glucosamine, mannosamine or galactosamine, or pentoses suchas arabinose, xylose or ribose.

Examples of the oligosaccharides are disaccharides such as maltose,lactose, trehalose, cellobiose, isomaltose, gentiobiose, melibiose,laminaribiose, chitobiose, mannobiose or sophorose; or maltotriose,isomaltotriose, taltotetraose, maltopentaose, mannotriose ormanninotriose.

The polymers or copolymers containing glycoside derivatives disclosed inPCT Application No. 90/04598 can be used for the above method.

There is no limitation in the molecular weight of the polymer orcopolymer containing glycoside derivative if said polymer or copolymeris soluble in the reaction medium. A law molecular weight requires muchamount of polymers or copolymers, thus taking much time to dissolve themin the reaction medium. Therefore, the molecular weight is preferablymore than 3,000.

The concentration of the polymer or copolymer containing glycosidederivatives in the reaction system of the antigen-antibody reaction issuitably determined depending upon the molecular weight of the polymeror copolymer, coexisting additives such as salts, proteins, orsaccharides. Generally, the polymer or copolymer is adjusted to becontained in the reaction system in an amount such that the finalconcentration at the time of reaction is 0.01 to 10.0% (W/V), preferablyabout 0.1 to 5.0% (W/V), more preferably about 0.5 to 2.0% (w/V). Whenthe concentration of the above polymer or copolymer is less than 0.01%(W/V), the above polymer or copolymer is less effect for acceleratingthe antigen-antibody reaction. On the other hand, the polymer orcopolymer having more than 10.0% (w/v) of the concentration increases innonspecific reaction with the materials other than the objectconstituents.

The present invention will be explained in detail hereinbelow withreference to Examples, by which no limitation shall not be given.

EXAMPLE 1 Method for purifying antigen

TP antigens were purified according to a method of the presentinvention.

1. Reagents and ethers

(1) Buffer solution

(1-1) Phosphate buffered saline (pH:6.5) (hereinafter abbreviated toPBS)

A buffer was prepared from potassium dihydrogen phosphate, sodiumdihydrogen phosphate (12 hydrate) and sodium chloride to make phosphateconcentration of 0.036M, NaCl concentration of 0.156M and pH 6.5. Tothis buffer was added NaN₃ at a concentration of 0.1% (w/v).

(1-2) 1% BSA/PBS

Bovine serum albumin (hereinafter abbreviated to BSA, manufactured byMiles Laboratories Co.) was dissolved in PBS to make 1% (W/V) BSAsolution. (1-3) 10 mM potassium phosphate buffer

(pH:6.0 and pH:7.0) (hereinafter abbreviated to KPB)

10 mM potassium dihydrogen phosphate solution was mixed with 10 mMdipotassium hydrogen phosphate to obtain potassium phosphate buffershaving pH 6.0 and 7.0.

(1-4) 350 mM KPB (pH:6.0)

350 mM potassium dihydrogen phosphate was mixed with 350 mM dipotassiumhydrogen phosphate to obtain mM potassium phosphate buffer having pH6.0.

(2) Surfactant

Octylglucopyranoside (1-O-n-octyl-β-D-glucopyranoside (hereinafterabbreviated as OG) used for a study of slightly soluble proteins(manufactured by Nacalai Tesque, Inc.) was employed as a surfactant.

(3) Chromatographic gel for purifying protein

(3-1) Cation exchanger

Sepharose Fast Flow (Pharmacia LKB Biotechnology), which is cationexchanger wherein a sulfonic acid group was introduced to the surface ofagarose gel, was used as cation exchanger.

(3-2) Hydroxyapatite gel

Bio-Gel® HTP (Bio-Rad Laboratories) and HCA-200L (Mitsui ToatsuChemicals, Inc.) were used as hydroxyapatite gel.

(4) TP

Treponemes were used was cultivated and isolated by the followingmethod.

A suspension of a pathogenic standard Nichols strain of Treponemapallidum (6.0×10⁷ /ml) was inoculated in rabbit testes in an amount of 1ml per testis. After the cultivation for 10 days, testes were taken outfrom 10 rabbits, sliced and then shaked for 30 minutes at 37° C. in 2.2%sodium citrate solution (500 ml). Thereafter, proliferated treponemeswere extracted. The extract was centrifuged for 5 minutes at 200×g toremove the precipitate of rabbit tissue. The supernatant was centrifugedfor 30 minutes at 3000×g to precipitate treponemes. Thus obtainedtreponemes were well washed with PBS and suspended in PBS to adjust thenumber of the treponemes to 1×10⁹ after the counting with a dark-fieldmicroscope. Thus, a suspension of TP mycelia was obtained. Thissuspension was confirmed with a dark-field microscope that no sperms andtissues of rabbit were included.

(5) Reagent for measuring concentration of protein

BCA® Protein Assay Reagent (Pierce Co.) was used as a reagent formeasuring concentration of protein.

(6) Microtiter plate

A microplate having 96 wells (Nunc Co., U-bottom) was used.

(7) TP antigen sensitized erythrocyte

Used sensitized erythrocyte was the one used for Seroclit TP (The Chemo-Sero-Therapeutic Research Institute), which is a TPHA kit on market.

(8) Syphilis-positive serum of rabbit

Used serum was the one taken out from the rabbit which was subjected tothe cultivation of TP in its testes for 45 days. Antibody titer wasmeasured by the commercially available TPHA kit, obtaining a value of102,400. This serum was diluted with 1% BSA/PBS for use.

2. Experimental Method

(2-1) Solubilization and extraction of antigen from TP

The suspension of treponemes (10 ml) was washed three times with PBS (50ml), followed by suspending in PBS (20 ml). The resultant suspension wassonicated for disruption. The suspension was subjected to centrifugationfor 30 minutes at 12,600×g to take out the precipitate.

Thus obtained precipitate was washed twice with KPB (10 mM, pH 7.0)using centrifugation for 30 minutes at 12,600×g. Thereafter, KPB (10 mM,pH 7.0) containing OG in 1% (W/V) was added in an amount of 25 ml to theprecipitate. The suspension was then slightly sonicated to besolubilized. After being left at 4° C. for 16 hours or more, the mixturewas centrifuged for 1 hour at 50,000×g. The resulting supernatant wasfiltered through a filter of 0.22 μm (Millex-GS produced by MilliporeCorporation). The extract obtained from the treponemes in this way wasreferred to as the extracted antigen hereinbelow.

(2-2) Pretreatment

(1) Dialysis of antigen solution

The extracted antigen which was dissolved in a buffer having pH 7.0 wasdialyzed against a buffer having pH 6.0 and containing 1% OG. Thedialysis was carried out with the volume ratio of the dialyzing solutionto the extracted antigen solution being 4 to 1. The dialyzing solutionwas exchanged three times. After the final dialysis, the pH value of thedialyzing solution (external solution for dialysis) was confirmed to bein the range of from 6.0±0.1.

(2) The antigen obtained in (1) was passed through a column (PharmaciaLKB Biotechnology, SR 25/45) of S Sepharose Gel (30 ml) to collect 50 mlof the passed-through fraction as the antigen fraction (hereinbelowreferred to as the partially purified antigen).

(2-3) Purification of antigen by hydroxyapatite gel

(1) Washing of hydroxyapatite gel

A column (Pharmacia LKB Biotechnology, HR 10/10) was filled with thehydroxyapatite gel (8 ml), followed by equilibrated with 10 mM KPBcontaining 1% OG (pH 6.0). The optical density (hereinafter abbreviatedto O.D.) of the washing solution at 280 nm was measured. Washing wascontinued until the absorbance of the eluate decreases to 0.010.

(2) Addition of the partially purified antigen

The partially purified antigen was added to a hydroxyapatite column.Thereafter, 10 mM KPB containing 1% OG (pH 6.0) was passed through thecolumn. The column was washed until the absorbance of the eluatesolution becomes 0.010 or less at 280 nm. The fraction thus obtained wasdefined as the passed-through fraction.

(3) Elution of antigen

A linear gradient elution was conducted by gradually increasing theratio of 350 mM KPB containing 1% OG to 10 mM KPB containing 1% OG (pH6.0) from 0 to 40%, and finally increasing it to 100% The fractions ofthe ratios of 0-8%, 8-16%, 16-24% 24-32% and 32-40% were collected. Thefraction at the ratio of 100% is defined as the 40% or more fraction.

(4) Assay of each fraction

The antigen activity and protein concentration of each of the extractedantigen, the passed-through fraction, 0-8% fraction, 8-16% fraction,16-24% fraction, 24-32% fraction, 32-40% fraction and 40% or morefraction were measured by the following methods. Specific activitiy wascalculated from the antigen activity and protein concentration.

(2-4) Antigen assay method

(1) Protein concentration

The protein concentration was measured by BCA® protein Assay Reagent(Pierce Co.) in which a measuring method [Smith, P. K., Krohn, R. I.etc., (1985) Anal. Biochme. 150, 76-85] was used as its principle. Saidmeasuring method uses bicinchoninic acid and is one of the modifiedLawry method. The used standard was a solution of BSA in 10 mM KPBcontaining 1% OG. The unit was expressed by μg/ml.

(2) Antigen activity

(a) 25 μl of 1% BSA/PBS was dispersed in each well of the microtiterplate.

(b) 25 μl of each antigen fraction was dispersed in the well of theplate and 25 μl of it was transferred in the above next well repeatedlyto be diluted serially 2¹ to 2^(n) fold with 1% BSA/PBS on the plate.

(c) Syphilis-positive rabbit serum, in which the antibody titer wasdiluted to 50 fold, was added to and mixed with the antigen fractionsdiluted 2¹ to 2^(n) fold in (b).

(d) The mixture was incubated for 30 minutes or more at roomtemperature. The antibody in the well having a high concentration ofantigen was consumed by antigen-antibody reaction, while the antibody onthe well having a low concentration of antigen remained thereon.

(e) Subsequently, TP antigen sensitized erythrocyte in the commerciallyavailable TPHA kit (Serodia-TP) was added to each well. A final dilutionratio of the rabbit serum which caused the hemagglutination was definedas the antigen activity. This antigen activity was represented by titer(titer/ml).

(g) Among the fractions exhibiting the antigen activity, thoseexhibiting a specified activity of 12 titer/μg or more were collected.Thus collected fractions were concentrated under reduced pressure byusing cellophane tube (Wako Pure Chemical Industries, Ltd.) until theprotein concentration became to 50 μg/ml or more. The obtained fractionswere defined as the HAp purified antigen.

3. Other experiments

Antigens were purified with Bio-Gel® HTP and HCA-200 L by the samemanner as described above. Besides the antigen activity and proteinconcentration of each of the fractions, those of extracted antigen andpartially purified antigen were measured, whereby total antigenactivity, total amount of protein and total specific activity of antigenwere calculated. Table 1 shows the results.

4. Conclusion

As is apparent from Table 1, when the TP extract was eluted afteradsorbing on the hydroxyapatite gel, the antigens were eluted fromBio-Gel® HTP at a salt concentration of 8% (37.2 mM) to 40% (146.0 mM)and from HCA-200 L at a salt concentration of 8% (37.2 mM) to 32% (118.8mM). Thus obtained fractions were combined, thereby obtaining the TPantigen of high purity having specific activities of 26.6 and 36.7(titer/pg) respectively.

                  TABLE 1-(1)                                                     ______________________________________                                        Purification from Bio-Gel ® HTP                                                                             Specified                                              Total Antigen                                                                             Total amount                                                                             Activity                                               Activity    of Protein of Antigen                                  Fractions  (titer)     (μg)    (titer/μg)                               ______________________________________                                        passed-through                                                                             0         850        --                                           0-8%        0          90        --                                           8-16%     4610        170        27.1                                        16-24%     9220        240        38.4                                        24-32%     4610        180        25.6                                        32-40%     2300        190        12.1                                        40% or more                                                                                0         430        --                                          Total of 8-40%                                                                           20740       780        26.6                                        Extracted antigen                                                                        24000       4250        5.6                                        Partially purified                                                                       24000       2370       10.1                                        antigen                                                                       ______________________________________                                    

                  TABLE 1-(2)                                                     ______________________________________                                        Purification from HCA-200L                                                                                      Specified                                              Total Antigen                                                                             Total amount                                                                             Activity                                               Activity    of Protein of Antigen                                  Fractions  (titer)     (μg)    (titer/μg)                               ______________________________________                                        passed-through                                                                             0         760        --                                           0-8%        0         110        --                                           8-16%     2300        120        12.1                                        16-24%     9220        140        65.0                                        24-32%     4610        180        25.0                                        32-40%      580        360         1.6                                        40% or more                                                                                0         700        --                                          Total of 8-40%                                                                           16130       440        36.7                                        Extracted antigen                                                                        24000       4250        5.6                                        Partially purified                                                                       24000       2370       10.1                                        antigen                                                                       ______________________________________                                    

Reference Example 1 Confirmation of protein purity by SDS-PAGE

1. Materials

(1) Electrophoretic apparatus

Phastsystem (Pharmacia) was used in accordance with its instruction.

(2) Molecular weight marker

Used molecular weight marker was the LMW kit E manufactured by PharmaciaLKB Biotechnology.

(3) Buffer for treating sample

The buffer was prepared by adding sodium dodecyl sulfate (5%) andmercaptoethanol (10%) to a solution of 10 mM Tris-HCl and 2 mM EDTA (pH8.0).

(4) Polyacrylamide gel

PhastGel Gradient 10-15 (Phast System) was used.

(5) Staining solution

A high-sensitive argentation solution for electrophoresis "Sil-BestStain for Protein/PAGE" (Nacalai Tesque, Inc.) was used in accordancewith the instruction.

2. Operation Method

(1) Preparation of samples

The extracted antigen, partially purified antigen or HAp purifiedantigen as obtained in Example 1 were mixed with the sample buffer in anequal amount and incubated for 5 minutes at 100° C.

(2) Preparation of the molecular weight marker

The molecular weight marker was dissolved in the sample buffer which wasdiluted in two fold with purified water, and then the solution wasincubated for 5 minutes at 100° C.

(3) The above-mentioned samples were loaded on the polyacrylamide gel inan amount of 1 μl for electrophoresis.

(4) After electrophoresis, the polyacrylamide gel was stained and themolecular weight of protein was calculated from its position in the gel.

3. Result

About 20 bands were observed in the extracted antigen, while about 10and 3 bands (at molecular weights of about 31,000, 41,000 and 47,000)were observed in the partially purified antigen and HAp purified antigenrespectively.

4. Conclusion

As is apparent from the result, an extremely high-purified TP antigenfraction was obtained by using the hydroxyapatite gel.

EXAMPLE 2 TPHA

The purified antigen obtained in accordance with the present inventionwas carried on sheep erythrocyte for confirming the effect of theinvention by TPHA.

1. Materials

The same materials as used in Example 1 were used if unspecified. Thebuffer was prepared by the same manner as in Example 1.

(1) Buffer

(a) 0.15M sodium phosphate buffer (pH:7.4):

The buffer was prepared by mixing 0.15M sodium dihydrogen phosphate (2hydrate) with 0.15M disodium hydrogen phosphate (12 hydrate) so as toshow pH 7.4.

(b) Physiological saline solution

The saline solution was prepared by dissolving sodium chloride (9.0 g)in purified water (1000 g).

(c) McIlvaine buffer (hereinafter abbreviated to McI)

McI was prepared by mixing 0.10M citric acid with 0.20M disodiumhydrogen phosphate (12 hydrate) so as to show pH 6.5.

(d) 1% OG/McI

The above-identified solution was obtained by dissolving OG (1% W/V) inMcI.

(e) PHA buffer

The above-identified buffer was prepared by mixing the solutions andreagents described as follows (The amount is expressed per 1000 ml ofbuffer.)

    ______________________________________                                        Rabbit normal serum     30     ml                                             Sheep erythrocyte stroma                                                                              10     ml                                             Sodium azide            1      g                                              0.15M sodium phophate buffer                                                                          100    ml                                             (pH 7.4)                                                                      Physiological saline    860    ml                                             ______________________________________                                    

(2) Reagents

(a) Tannic acid was bought from Nacalai Tesque, Inc.

(b) Fixed sheep erythrocyte was the one immobilized with glutaraldehyde.

(c) TPHA kits commercially available

Serodia TP (Fuji Rebio Inc.) and Seroclit TP (The Chemo-Sero-Therapeutic Research Institute) were used.

(3) Serum Samples

(a) Syphilis-positive control

Three control sera (G₁, G₂ and G₃) were used, those of which werecollected from fully cured advanced syphilitic patients. These controlsera were supposed to contain a lot of IgG antibodies. Further, threecontrol sera (M₁, M₂ and M₃) were used, those of which were collectedfrom primary syphilitic patients, i.e., three to five weeks after theinfection. The latter controls were supposed to contain few Ig-Gantibodies, but Ig-M antibodies.

(b) Normal control (Syphilis-negative control)

Three control sera (N₁, N₂ and N₃) were used, those of which wereobserved to show nonspecific reaction with the TPHA kit commerciallyavailable and further found not to be syphilic by FTA-ABS.

(c) Anti-rabbit tissue antiserum

A normal rabbit testis sliced and solubilized with 1% OG was immunizedto a goat for obtaining the above-identified serum.

(d) Anti-Reiter strain antiserum

Nonphthogenic treponemes, Treponema phagedenis (biotype Reiter), weresolubilized with 1% OG. This solution was immunized to a goat forobtaining the above-identified serum.

(4) TP antigen

The extracted antigen, partially purified antigen and HAp purifiedantigen as obtained in Example 1 were used. Table 2 shows the antigenactivity and protein concentration of each antigen solution.

                  TABLE 2                                                         ______________________________________                                                                            Specified                                             Antigen Activity                                                                           Protein cocn.                                                                            Activity                                  Antigen     (titer/ml)   (μg/ml) (titer/μg)                             ______________________________________                                        Extracted Antigen                                                                         2048         365         5.6                                      Partially purified                                                                        1024         101        10.1                                      antigen                                                                       (HAp Purification)                                                            Bio-Gel ® HTP                                                                         2048         77.0       26.6                                      HCA-200L    1536         41.9       36.7                                      ______________________________________                                    

2. Method

(1) Blood cell processing method

(a) The fixed sheep blood cells were washed four times with aphysiological saline by using centrifugation for 5 minutes at 700×g,which was suspended in a physiological saline to have solid content of6%. A solution (tannic acid in physiological saline, 120 μg/ml) wasadded to the blood cell suspension, followed by stirring.

(b) After stirring, the resultant solution was washed twice with aphysiological saline and once with McI, and then suspended in McI tohave solid content of 6%. Immediately, the resultant solution was usedfor antigen sensitization.

(c) TP antigen solution (the extracted antigen solution, partiallypurified antigen solution or HAp purified solution) was in advancedialyzed against 1% OG/McI. The obtained solution was adjusted asdescribed in Table 3 to serve as the sensitizing solution. Thesensitizing solution A was adjusted to have antigen activity of 100(titer/ml). The sensitizing solution B was adjusted to have proteinconcentration of 10 μg/ml. One volume of the sensitizing solution wasadded to one volume of the blood cell suspension as described at (b) andthe mixture was stirred for 1 hour at 25° C.

                  TABLE 3                                                         ______________________________________                                                 Sensitizing Solution A                                                                     Sensitizing Solution B                                           (Sensitization with                                                                        (Sensitization with                                              a predetermined                                                                            a predetermined                                                  antigen amount)                                                                            protein amount)                                                    Antigen  1% OG/    Antigen                                                                              1% OG/                                              Solution McI       Solution                                                                             McI                                      Antigen    (ml)     (ml)      (ml)   (ml)                                     ______________________________________                                        Extracted  0.049    0.951     0.027  0.973                                    Partially purified                                                                       0.098    0.902     0.099  0.901                                    (HAp                                                                          purification)                                                                 Bio-Gel ® HTP                                                                        0.049    0.951     0.130  0.870                                    HCA-200L   0.065    0.935     0.239  0.761                                    ______________________________________                                    

(d) The sensitized blood cells were washed twice with a physiologicalsaline and suspended in PHA buffer to have blood cell solid content of0.2%. After standing for 3 hours at room temperature, the resultantsuspension was used for assay.

(2) TPHA assay method

(a) 100 μl of PHA buffer was dispensed in each well of the microtiterplate, and 25 μl in other wells.

(b) 25 μl of each control was dispensed in each well of the microtiterplate, which was serially diluted 2¹ to 2^(n) on the plate.

(c) The blood cell suspension prepared in (1) was shaken to obtain ahomogeneous suspension. 75 μl of the suspension was dispensed in eachwell.

(4) The plate was vibrated to mix sufficiently. Thereafter, the platewas covered with an empty plate in order to prevent evaporation and thenincubated at room temperature. The determination was conducted after 2hours.

(5) The plate was placed on white paper, observing hemagglutination byvisual observation. The maximum dilution (20, 40, 80, . . . ) exhibitingagglutination was defined as the antibody titer.

3. Result

Table 4 shows the results of determination of hemagglutination on eachcontrol and of measurement of antibody titer by using the sensitizedblood cell obtained by the above method.

As shown in Table 4, three primary syphilitic sera which were allnegative with the sensitized blood cell using the extracted antigen werepositive with the sensitized blood cell using the HAp purified antigenof the present invention.. Normal control sera exhibiting nonspecificreaction by the TPHA kit showed no agglutination. Further, thesensitized blood cell using the present invention exhibited noagglutination with the anti-rabbit tissue antiserum and anti-Reiterstrain-antiserum.

4. Conclusion

As is apparent from the result, even a primary syphilis is detectable byusing the antigen purified according to the present invention, withoutgiving a false positive result due to the nonspecific reaction.

                                      TABLE 4                                     __________________________________________________________________________    Result of TPHA                                                                                       Partially                                              Antigen  HAp purified antigen                                                                        purified                                                                           Extracted                                                                           Commercial                                  Control  Bio-Gel ® HTP                                                                     HCA-200L                                                                            antigen                                                                            antigen                                                                             TPHA                                        __________________________________________________________________________    (1) Sensitizing Solution (A) Sensitization with a predetermined antigen       amount                                                                        Primary                                                                             M1 +       +     ± -     -                                           syphilis ×160                                                                            ×160                                                                           ×80                                                                         ×40                                                                           ×40                                         M2 +       +     +    ±  ±                                                 ×320                                                                            ×320                                                                          ×160                                                                         ×80                                                                           ×80                                         M3 +       +     ± -     -                                                    ×160                                                                            ×160                                                                           ×80                                                                         ×40                                                                           ×40                                   Advanced                                                                            G1 +       +     +    +     +                                           syphilis ×1280                                                                           ×1280                                                                         ×1280                                                                        ×320                                                                          ×320                                        G2 +       +     +    +     +                                                    ×640                                                                            ×640                                                                          ×640                                                                         ×320                                                                          ×320                                        G3 +       +     +    ±  ±                                                 ×320                                                                            ×320                                                                          ×160                                                                         ×80                                                                           ×80                                   Normal                                                                              N1 -       -     -    -     -                                                     <20     <20   <20 <40   <40                                               N2 -       -     -    +     +                                                     <20     <20   ×40                                                                         ×160                                                                          ×160                                        N3 -       -     -    ±  ±                                                  <20     <20   ×20                                                                         ×80                                                                           ×80                                   Anti-rabbit                                                                            -       -     -    +     -                                           tissue antiserum                                                                        <20     <20   ×40                                                                         ×160                                                                          ×20                                   Anti-Reiter                                                                            -       -     -    +     -                                           strain antiserum                                                                        <20     <20   ×40                                                                         ×160                                                                          <20                                         __________________________________________________________________________    (2) Sensitizing solution (B) Sensitization with a predetermined protein       amount                                                                        Primary                                                                             M1 +       +     ± -     -                                           syphilis ×160                                                                            ×160                                                                           ×80                                                                         <20   ×40                                         M2 +       +     +    -     ±                                                 ×320                                                                            ×320                                                                          ×160                                                                         ×40                                                                           ×80                                         M3 +       +     ± -     -                                                    ×160                                                                            ×160                                                                           ×80                                                                         ×40                                                                           ×40                                   Advanced                                                                            G1 +       +     +    +     +                                           syphilis ×1280                                                                           ×1280                                                                         ×1280                                                                        ×320                                                                          ×320                                        G2 +       +     +    +     +                                                    ×640                                                                            ×640                                                                          ×640                                                                         ×160                                                                          ×320                                        G3 +       +     +    ±  ±                                                 ×320                                                                            ×320                                                                          ×160                                                                         ×40                                                                           ×80                                   Normal                                                                              N1 -       -     -    -     -                                                     <20     <20   <20 ×40                                                                           ×40                                         N2 -       -     -    ±  +                                                     <20     <20   ×40                                                                         ×80                                                                           ×160                                        N3 -       -     -    ±  ±                                                   <20    <20  ×20                                                                          ×80                                                                           ×80                                   Anti-rabbit                                                                            -       -     -    ±  -                                           tissue antiserum                                                                        <20     <20   ×40                                                                         ×80                                                                           ×20                                   Anti-Reiter                                                                            -       -     -    ±  -                                           strain antiserum                                                                        <20     <20  ×40                                                                          ×80                                                                           <20                                         __________________________________________________________________________     +: positive (antibody titer more than 80)                                     ±: false positive (antibody titer of 80)                                   -: negative (antibody titer less than 80)                                     The value shown below each result represents an antibody titer.          

EXAMPLE 3 Latex reagent (for measuring method using fully-automaticanalyzer)

In the case of detecting anti-treponemal antibodies, the effect of thediagnostic latex reagent as prepared by the present invention wasconfirmed by measuring the agglutination with the use of afully-automatic analyzer. Controls as those used in Example 1 andExample 2 were prepared by the same manner as in Example 1 and Example 2if unspecified.

1. Preparation of reagent and control

(1) Latex

Polystyrene latex (solid:10%) of 0.400 μm (Sekisui Chemical Co., Ltd.)was used.

(2) PBS (pH:7.4)

A solution of 0.02M phosphate and 0.15M sodium phosphate (2 hydrate),disodium phosphate (2 hydrate) and sodium chloride, to which NaN₃ (aspreservative) was added at 0.1%.

(3) NaCl--PBS (pH: 6.5)

A buffer was prepared from sodium phosphate (2 hydrates), disodiumphosphate (2 hydrates) and sodium chloride to make phosphateconcentration of 0.02M, NaCl concentration of 1.00M and pH 6.5.

(4) 100 mM NaPB

A solution of 100 mM NaPB (pH:7.5) were prepared from disodium hydrogenphosphate (anhydrous) and sodium dihydrogen phosphate (12 hydrate), towhich NaN₃ was added at 0.1%.

(5) 1% BSA--NaPB

The above-identified solution was prepared to have BSA in 100 mM NaPB.

(6) 5% BSA--NaPB

The above-identified solution was prepared to have 5% BSA in 100 mMNaPB.

(7) Diluent

The diluent was prepared by dissolving polyethylene glycol (averagemolecular weight:500,000, Wako Pure Chemical Industries, Ltd.) in 0.25%(W/V) in BSA--NaPB.

(8) Instrument

Measurement was performed on Hitachi 7050 Type, a fully-automaticanalyzer.

2. Method

(1) Preparation of antigen sensitizing solution

To each antigen solution having antigen titer and protein concentrationshown in Table 2 was added a mixture of 10 mM KPB, of NaCl--PBS and 1%OG in the amount shown in Table 5. The obtained solution was defined asthe sensitizing solution (the antigen solution for immobilization)having pH 5.4 to 6.5. The sensitizing solution A was prepared to haveantigen activity of 250 (titer/ml), while the sensitizing solution B wasprepared to have protein concentration of about 25 μg/ml.

                  TABLE 5                                                         ______________________________________                                        Sensitizing solution A                                                        (Sensitization with a predetermined antigen amount)                                       Antigen     1% OG/KPB  NaCl/PB                                    Antigen     Solution (ml)                                                                             pH 6.0 (ml)                                                                              (ml)                                       ______________________________________                                        Extracted antigen                                                                         0.049       0.251      0.100                                      Partially purified                                                                        0.098       0.202      0.100                                      antigen                                                                       (HAp purification)                                                            HCA-200L    0.049       0.251      0.100                                      Bio-Gel ® HTP                                                                         0.065       0.235      0.100                                      ______________________________________                                    

    ______________________________________                                        Sensitizing solution B                                                        (Sensitization with a predetermined protein amount)                                       Antigen     1% OG/KPB  NaCl/PB                                    Antigen     Solution (ml)                                                                             pH 6.0 (ml)                                                                              (ml)                                       ______________________________________                                        Extracted antigen                                                                         0.027       0.273      0.100                                      Partially purified                                                                        0.099       0.201      0.100                                      antigen                                                                       (HAp purification)                                                            HCA-200L    0.130       0.170      0.100                                      Bio-Gel ® HTP                                                                         0.239       0.061      0.100                                      ______________________________________                                    

(2) Immobilization of treponemal antigen

Polystyrene Latex (100 μl) (solid content of 10% by weight) was stirredby a magnetic stirrer in an incubator at 4° C., with which thetreponemal antigen solution (400 μl) prepared in (1) was quickly mixedand stirred for 1 hour at 4° C. After the addition of 1% BSA (5 ml), theresultant mixture was stirred for 1.5 hours at 4° C. and thencentrifuged for 1 hour at 15,000 rpm. To the obtained pellets was addedagain 1% BSA--NaPB (5 ml). The resultant mixture was centrifuged by thesame manner as described above and washed. 1% BSA--NaPB (5 ml) was addedto the final pellets and sufficiently dispersed, thereby affording latexreagent having solid content of 0.2%. Thus obtained Latex reagent waskept at 4° C.

(3) Parameters of Hitachi 7050 Type, a fully-automatic analyzer

    ______________________________________                                        Sample content    20 μl (serum)                                            R1 content        50 μl (Latex reagent)                                    R2 content        350 μl (diluent)                                         Wavelength        570 nm                                                      ______________________________________                                    

(4) Measuring method

The difference of the absorbance between 80 seconds after the beginningof the measurement and 320 seconds after the beginning of themeasurement was taken. 10⁴ of this difference was defined as thevariation at O.D. 570.

3. Result

The reaction of each control with the Latex reagent prepared from the TPantigen solution by the manner described above was measured as thevariation of turbidity at O.D. 570. Table 6 shows the results.

As shown in Table 6, three primary syphilitic sera which were allnegative with the latex reagents using the extracted antigen andpartially purified antigen exhibited sensitivity sufficient fordetermining to be positive with the latex reagent using the purified TPantigen of the present invention. Three advanced syphilitic seraexhibiting nonspecific reaction with the TPHA kit showed noagglutination with the latex reagent using the purified TP antigen ofthe present invention. Further, the latex reagent using the presentinvention did not exhibit a turbidity for determining to be positivewith the anti-rabbit tissue antiserum and anti-Reiter strain antiserum.

4. Conclusion

As is apparent from the result, the reagent prepared from the purifiedantigen according to the present invention is more reactive, i.e.,high-sensitive compared to there from the conventional extractedantigen. As a result, even primary syphilis, which cannot be detected bythe conventional extracted antigen can be detected by using the purifiedantigen according to the present invention.

Further, the use of the purified antigen can detect primary syphilis,which cannot be detected by the commercially available TPHA kit, andalso does not give the false positive result by the nonspecificreaction.

                  TABLE 6                                                         ______________________________________                                        Result with a latex reagent                                                            HAp purified antigen                                                                        Partially                                                                              Ex-                                           Antigen    Bio-Gel ®       purified                                                                             tracted                                 Control    HTP       HCA-200L  antigen                                                                              antigen                                 ______________________________________                                        Sensitizing Solution (A)                                                      Sensitization with a predetermined antigen amount                             Primary M1     323        639    157    44                                    syphilis                                                                              M2     857       1433    184    103                                           M3     646        626    108    38                                    Advanced                                                                              G1     2101      3289    1624   678                                   syphilis                                                                              G2     1655      2273    683    275                                           G3     849       1274    154    110                                   Normal  N1     4           2      19    55                                            N2     3           5      16    21                                            N3     2           7      19    21                                    Anti-rabbit    3           3      40    105                                   tissue                                                                        antiserum                                                                     Anti-Reiter    5           9      32    97                                    strain                                                                        antiserum                                                                     Sensitizing Solution (B)                                                      Sensitization with a predetermined protein amount                             Primary M1     1231      1710    161    16                                    syphilis                                                                              M2     3144      3882    171    41                                            M3     1231      1695    122    21                                    Advanced                                                                              G1     7812      7540    1585   291                                   syphilis                                                                              G2     6129      6148    652    100                                           G3     3102      3225    171    65                                    Normal  N1     5         3       317     5                                            N2     2         2        15     3                                            N3     3         7        19    10                                    Anti-rabbit    9         6        39    66                                    tissue                                                                        antiserum                                                                     Anti-Reiter    5         8        41    35                                    strain                                                                        antiserum                                                                     ______________________________________                                    

EXAMPLE 4 Assay for syphilis antibody by ELISA method

The effect of the purified antigen of the present invention wasconfirmed by ELISA method.

1. Preparation of reagent and control

The following reagents and controls were prepared for use. The samereagents and controls as those used in Examples 1, 2 and 3 were preparedby the same manner as in Examples 1, 2 and 3.

(1) TP antigen solution

Used antigen solution were the extracted antigen, partially purifiedantigen and HAp purified antigen obtained in Example 1.

(2) Control

The controls used in Example 2 were diluted by a hundred fold with 1%BSA/PBS.

(3) Peroxidase labelled anti-goat Ig-G

Peroxidase labelled anti-goat Ig-G (originated from sheep) (MilesLaboratories Co.) was diluted by a thousand fold with 1% BSA/PBS withoutNaN₃.

(4) Peroxidase labelled anti-human Ig-G and Ig-M

Peroxidase labelled anti-goat Ig-G and Ig-M (originated from sheep)(Miles Laboratories Co.) were diluted by a thousand fold with 1% BSA/PBSwithout NaN₃.

(5) Microtiter plate

A microtiter plate having 96 wells (Nunc Co., flat bottom for ELISA) wasused.

(6) Peroxidase substrate

o-Phenylenediamine (dihydrochloride) (2 mg/ml) and aqueous hydrogenperoxide (0.03%) were dissolved in a phosphoric acid-citric acid buffer(pH 5.0). The substrate was prepared immediately before being used.

(7) Stop solution

1N sulfuric acid solution was used as the stop solution of enzymereaction.

2. Method

(1) Preparation of antigen solution

By the same manner as in Example 3, to each antigen solution was added amixture of 10 mM KPB, NaCl--PB and 1% OG in the amount shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        (Composition of sensitizing solution for ELISA)                                          Antigen                                                                       Solution  1% OG/KPB   NaCl/PBS                                     Antigen    (ml)      (ml)        (ml)                                         ______________________________________                                        Sensitizing solution A                                                        (Sensitization with a predetermined antigen amount)                           Extracted  0.049     0.351       0.100                                        antigen                                                                       Partially  0.098     0.302       0.100                                        purified                                                                      antigen                                                                       (HAp                                                                          purification)                                                                 HCA-200L   0.049     0.351       0.100                                        Bio-Gel ® HTP                                                                        0.065     0.335       0.100                                        Sensitizing solution B                                                        (Sensitization with a predetermined protein amount)                           Extracted  0.027     0.373       0.100                                        antigen                                                                       Partially  0.099     0.301       0.100                                        purified                                                                      antigen                                                                       (HAp                                                                          purification)                                                                 HCA-200L   0.130     0.270       0.100                                        Bio-Gel ® HTP                                                                        0.239     0.161       0.100                                        ______________________________________                                    

(2) Immobilization of the TP antigen

The TP antigen solution prepared in (1) was dispersed into themicrotiter plate in an amount of 50 μl and incubated for 1 hour at roomtemperature.

After the incubation, the excess TP antigen solution was removed andwashed three times with 1% BSA/PBS (200 μl) under suction. Thereafter,1% BSA/PBS (200 μl) was added to the resultant and incubated for 1 hourat room temperature for effecting blocking. The plate to which blockingwas completed was used for antigen-antibody reaction.

(3) Antigen-antibody reaction

As a first antibody, the control diluted hundredfold with 1% BSA/PBS waspipetted into each well in an amount of 100 μl . As a control, thecontrol was similarly pipetted into each well to which 1% BSA/PBS wasadded instead of the antigen. After incubated for 1 hour at roomtemperature, the solution was removed under suction and washed threetimes with 1% BSA/PBS (200 μl) under suction.

Then, the peroxidase labelled anti-human Ig-G and anti-human Ig-M werepipetted in an amount of 100 μl into each well into which the primarysyphilis sera, advanced syphilis sear and syphilis-negative sear werepipetted. Further, the anti-goat Ig-G was pipetted in an amount of 100μl into each well into which the anti-Reiter strain antiserum andanti-rabbit tissue antiserum were pipetted. After the incubation for 1hour at room temperature, each well was sucked and washed three timeswith 200 μl of 1% BSA/PBS. Immediately after washing, enzyme activitybound to each well was measured.

(4) Enzymatic reaction

100 μl of peroxidase substrate was added to each well and the plate wasincubated for 15 minutes at room temperature. As a substrate blank, thesubstrate was pipetted into each well not containing antigen, firstantibody or second antibody. After the incubation, 1N stop solution (100μl) was added to stop the enzymatic reaction. After stopping thereaction, the absorbance at 492 nm was measured with a microtiter platereader (MTP-100, Corona Electric Co., Ltd.), in comparison with thesubstrate blank.

3. Result

Table 8 shows the results of the absorbance at 492 nm.

Three primary syphilitic sear increase in detection sensitivity of IgMby using the purified antigen of the present invention. Threesyphilis-negative controls exhibit falsely positive (the value of morethan O.D. 0.057 is determined to be positive) by using the extractedantigen, while do not exhibit positive by using the purified antigen ofthe present invention.

Moreover, the purified antigen of the present invention did not show avalue for determining to be positive with the anti-rabbit tissueantiserum and anti-Reiter strain antiserum.

4. Conclusion

As is apparent from the above result, the purified antigen according tothe present invention can detect even primary syphilis, which can not bedetected by ELISA method using the conventional extracted antigen, andalso does not give a false positive result by the nonspecific reaction.

                                      TABLE 8                                     __________________________________________________________________________    Result of ELISA                                                                          HAp purified antigen                                                                       Partially                                                        Bio-Gel ®                                                                              purified                                                                            Extracted                                       Antigen    HTP    HCA-200L                                                                            antigen                                                                             antigen                                         Control                                                                             1st  anti                                                                              anti                                                                             anti                                                                             anti                                                                             anti                                                                             anti                                                                             anti                                                                             anti                                               antibody                                                                           Ig-G                                                                              Ig-M                                                                             Ig-G                                                                             Ig-M                                                                             Ig-G                                                                             Ig-M                                                                             Ig-G                                                                             Ig-M                                         __________________________________________________________________________    (1) Sensitizing Solution (A)                                                  Sensitization with a predetermined antigen amount                             Primary                                                                             M1   0.125                                                                             0.452                                                                            0.182                                                                            0.518                                                                            0.123                                                                            0.257                                                                            0.050                                                                            0.030                                        syphilis                                                                            M2   0.098                                                                             0.258                                                                            0.082                                                                            0.210                                                                            0.102                                                                            0.220                                                                            0.056                                                                            0.062                                              M3   0.058                                                                             0.142                                                                            0.064                                                                            0.264                                                                            0.045                                                                            0.084                                                                            0.057                                                                            0.042                                        Advanced                                                                            G1   0.984                                                                             0.245                                                                            0.845                                                                            0.214                                                                            0.789                                                                            0.145                                                                            0.874                                                                            0.168                                        syphilis                                                                            G2   1.489                                                                             0.478                                                                            1.378                                                                            0.347                                                                            1.540                                                                            0.210                                                                            1.554                                                                            0.355                                              G3   0.258                                                                             0.154                                                                            0.321                                                                            0.148                                                                            0.265                                                                            0.081                                                                            0.291                                                                            0.067                                        Negative                                                                            N1   0.015                                                                             0.014                                                                            0.012                                                                            0.016                                                                            0.032                                                                            0.024                                                                            0.064                                                                            0.056                                        syphilis                                                                            N2   0.020                                                                             0.016                                                                            0.009                                                                            0.013                                                                            0.051                                                                            0.024                                                                            0.224                                                                            0.095                                              N3   0.021                                                                             0.012                                                                            0.013                                                                            0.016                                                                            0.042                                                                            0.032                                                                            0.102                                                                            0.081                                        Anti-rabbit                                                                              0.009                                                                             -- 0.015                                                                            -- 0.104                                                                            -- 0.278                                                                            --                                           tissue                                                                        antiserum                                                                     Anti-Reiter                                                                              0.015                                                                             -- 0.012                                                                            -- 0.069                                                                            -- 0.225                                                                            --                                           strain                                                                        antiserum                                                                     (2) Sensitizing solution (B)                                                  Sensitization with a predetermined protein amount                             Primary                                                                             M1   0.138                                                                             0.513                                                                            0.173                                                                            0.476                                                                            0.138                                                                            0.289                                                                            0.042                                                                            0.046                                        syphilis                                                                            M2   0.082                                                                             0.376                                                                            0.099                                                                            0.410                                                                            0.132                                                                            0.253                                                                            0.043                                                                            0.051                                              M3   0.043                                                                             0.227                                                                            0.061                                                                            0.232                                                                            0.035                                                                            0.075                                                                            0.045                                                                            0.057                                        Advanced                                                                            G1   0.965                                                                             0.233                                                                            0.846                                                                            0.256                                                                            0.822                                                                            0.109                                                                            0.278                                                                            0.078                                        syphilis                                                                            G2   1.625                                                                             0.489                                                                            1.478                                                                            0.512                                                                            1.579                                                                            0.224                                                                            0.456                                                                            0.125                                              G3   0.289                                                                             0.167                                                                            0.335                                                                            0.154                                                                            0.258                                                                            0.084                                                                            0.085                                                                            0.054                                        Negative                                                                            N1   0.013                                                                             0.012                                                                            0.009                                                                            0.006                                                                            0.045                                                                            0.030                                                                            0.102                                                                            0.048                                        syphilis                                                                            N2   0.016                                                                             0.013                                                                            0.015                                                                            0.008                                                                            0.051                                                                            0.014                                                                            0.152                                                                            0.054                                              N3   0.017                                                                             0.018                                                                            0.016                                                                            0.006                                                                            0.038                                                                            0.031                                                                            0.106                                                                            0.043                                        __________________________________________________________________________    Anti-rabbit                                                                         0.014                                                                              --  0.012                                                                            -- 0.067                                                                            -- 0.156                                                                            --                                              tissue                                                                        anti-serum                                                                    Anti-Reiter                                                                         0.009                                                                              --  0.008                                                                            -- 0.078                                                                            -- 0.115                                                                            --                                              strain                                                                        antiserum                                                                     __________________________________________________________________________     Average value at O.D. 492 nm (n = 4)                                     

Reference Example 2 Confirmation of antigen protein by immunoplottingassay

1. Reagent and sample

The same reagents and samples as those used in Example 1, ReferenceExample 1 and Example 4 were prepared by the same manner as theseExamples.

(1) Buffer for blotting

The buffer was prepared by mixing 25 mM Tris buffer, 192 mM glycine and20% methanol to have pH 8.3.

(2) Nitrocellulose membrane (hereinafter abbreviated to NC)

Used membrane was 9×12 cm sheet having a pore size of 0.45μ (Bio-RadLaboratories).

(3) Syphilis-positive serum

Pooled primary syphilitic serum and advanced syphilitic serum were used.

(4) Peroxidase substrate

4-Chloro-1-naphtol (Nacalai Tesque Inc.) (10 mg) was dissolved inice-cooled methanol (3.34 ml) and then mixed with citrate phosphatebuffer (pH:6.0, 16.66 ml). To the resultant mixture was added 30%aqueous hydrogen peroxide (10 μl) immediately before being used. Thesubstrate was used immediately after preparation.

2. Operation method

(1) Phast System® (Pharmacia LKB Biotechnolgy) was used in accordancewith its instruction. Electrophoresis of the extracted antigen,partially purified antigen and HAp purified antigen was conducted by thesame manner as in Example 2. The same five gels obtained by theelectrophoresis were prepared.

(2) NC were in advance immersed in the buffer for blotting to beequilibrated. The NC were pleased on the gel for transferring proteinfrom the gel to NC by electrophoresis with blotting.

(3) After washing three times with PBS, NC were immersed in PBS buffercontaining 3% BSA for blocking.

(4) The primary syphilitic serum, advanced syphilitic serum, normal(syphilis-negative) serum, anti-rabbit tissue antiserum and anti-Reiterstrain antiserum were diluted hundred fold with 1% BSA/PBS. Each serumwas reacted with each antigen transferred onto the above-mentioned NC.

(5) After washing three times with 1% BSA/PBS, the NC reacted with theprimary syphilitic serum, advanced syphilitic serum and normal serumwere immersed in 1% BSA/PBS containing the peroxidase labelledanti-human Ig-G and anti-human Ig-M for reaction. Similarly, the NCreacted with the anti-Reiter strain antiserum and anti-rabbit tissueantiserum were immersed in 1% BSA/PBS containing the anti-goat Ig-Gconjugated with peroxidese for reaction. After the incubation for 1 hourat room temperature, the NC were washed three times with PBS, andimmediately after that, enzyme activity bounded to each NC was observed.

(6) Peroxidase substrate was added to the NC and incubated at roomtemperature. When a suitable colored image was observed, the membraneswere washed with purified water and dried. Thereafter, the position anddegree of the color development were observed.

3. Result

It was confirmed that three bands (at the vicinity of the molecularweights 31,000, 41,000 and 47,000) of the HAp purified antigen wereproteins specifically reactive with syphilis-positive serum only. Thebands reacted with the anti-rabbit tissue-antiserum and anti-Reiterstrain antiserum were observed in the extracted antigen and partiallypurified antigen, while such bands were not found in the HAp purifiedantigen.

4. Conclusion

As is apparent from the result, the TP purified antigen obtained byusing the hydroxyapatite gel is a high-pure antigen solution consistingof the component specifically reactive with the syphilis-positive serumonly.

EXAMPLE 5 Conditions for adsorption to and elution by hydroxyapatite gel

The partially purified antigen solution obtained in Example 1 was used.Before adding to a column, the buffer of the partially purified antigensolution was exchanged to the buffer for adsorption by dialysis.

Bio-Gel® HTP (Bio-Rad) was used as hydroxyapatite gel.

Other reagents and conditions and methods for chromatography are thesame as those in Example 1.

Antigen purity (specific activity) in the obtained fractions wereobserved by varying the conditions for adsorption and elution by use ofBio-Gel®HTP. Table 9 shows the result.

                  TABLE 9                                                         ______________________________________                                        Buffer                                                                        Solution A  Solution B                                                        (Adsorption)                                                                              (Elution)     Fraction  Specific                                       phosphate         Phosphate                                                                              (ratio of                                                                             Activity                              pH   conc.      pH     conc.    B, %)   (titer/μg)                         ______________________________________                                        5.5  10 mM      5.5    350 mM   15-50   27.5                                  6.0  10 mM      6.0    350 mM   8-40    28.4                                  6.5   5 mm      6.5    350 mM   2-20    20.3                                  7.0   5 mM      7.0    350 mM   1-5     17.2                                  6.0  10 mM      8.0     10 mM   10-40   19.7                                  ______________________________________                                         note: Solution A and B are potassium phosphate buffer containing 1% OG.  

EXAMPLE 6 Latex reagent

The partially purified antigen solution obtained in Example 1 was usedwhich was dissolved in 10 mM potassium phosphate buffer (pH:6.0)containing 1% OG. Sensitizing solution having protein concentration of30μg/ml, predetermined OG concentration and pre-determined pH wasprepared by diluting the antigen solution with buffer or adding OG tothe antigen solution.

Latex reagent was prepared by the same manner as in Example 3.

Used control was obtained by diluting rabbit serum with phosphatebuffered saline [0.02M phosphate buffer, 0.13M sodium chloride (pH:7.4)and 0.1% sodium azide] containing 1% BSA.

The controls were measured with the obtained latex reagent by the samemanner as in Example 3. Table 10 shows the result.

                  TABLE 10                                                        ______________________________________                                        Sensitizing solution                                                                      % for       Variation                                             cocn. of        dilution    syphilis-                                                                            syphilis-                                  OG (%)    pH    (%)         negative                                                                             positive                                   ______________________________________                                        0.5       5.0   400         8.0    205.2                                                      200         6.0    423.0                                                      100         7.0    1024.0                                     0.5       6.0   400         3.0    237.0                                                      200         5.0    481.2                                                      100         13.0   1312.0                                     0.5       7.0   400         10.0   225.0                                                      200         9.0    453.6                                                      100         7.0    1200.0                                     0.5       7.5   400         4.0    186.6                                                      200         7.0    372.0                                                      100         12.0   960.0                                      0.025     6.0   400         6.0    198.0                                                      200         11.0   420.0                                                      100         10.0   1200.0                                     1.0       6.0   400         7.0    177.0                                                      200         8.0    372.0                                                      100         6.0    1088.0                                     2.0       6.0   400         3.0    192.0                                                      200         6.0    378.0                                                      100         9.0    1016.0                                     ______________________________________                                         note:                                                                         Controls are syphilisnegative and syphilispositive (10,000 titer) rabbit      serum.                                                                        Four hundred fold, two hundred fold and one hundred fold the                  syphilispositive serum respectively correspond to 25, 50 and 100 titer.  

What is claimed is:
 1. A process for preparing a purified syphilisantigen which comprises adsorbing an extract originated from Treponemapallidum on a hydroxyapatite gel, eluting with an aqueous mediumcontaining octylglucopyranoside and recovering the purified syphilisantigen.
 2. The process of claim 1, in which the adsorption is conductedwith an aqueous medium having a pH of 5.0 to 8.0 and an ionic strengthof 2 to 30 mM.
 3. The process of claim 1, in which the elution isconducted with an aqueous medium having a pH of 5.0 to 11.0 and an ionicstrength of 10 to 360 mM.
 4. The process of claim 1 or 2, in which theelution is conducted with an aqueous medium having either a pH or anionic strength which is higher than the pH or the ionic strength of theaqueous medium used for the adsorption.
 5. The process of claim 1 inwhich the aqueous medium is phosphate buffer.
 6. The process of claim 1in which the elution is conducted with a linear gradient.